Humanized Mice
T cells play a central role in the development of immune responses. Patients lacking T cells due to genetic defects such as DiGeorge or Nezelof’s syndrome and patients infected with the human immunodeficiency virus are highly susceptible to infections and cancers. The lack of adequate in vivo models of T cell neogenesis have hindered the development and clinical implementation of effective therapeutic modalities aimed at treating these and other clinically important maladies. Transplantation of severe combined immunodeficient (SCID) mice with human hematopoietic stem cells results in long-term engraftment and systemic reconstitution with human progenitor, B and myeloid cells but curiously, human T cells are rarely present in any tissue. While the implantation of SCID mice with human fetal thymus and liver (SCID-hu thy/liv mice) allows for the development of abundant thymocytes that are localized in the human organoid implant, there is minimal systemic repopulation with human T cells. We have shown that transplantation of autologous human hematopoietic fetal liver CD34+ cells into NOD/SCID mice previously implanted with fetal thymic and liver tissues results in long term, systemic human T cell homeostasis. In addition to human T cells, these mice have systemic repopulation with human B cells, monocytes/macrophages and dendritic cells (DC). Importantly, the T cells developed in these mice are capable of being activated by human DCs and mount potent T cell immune responses to toxic shock syndrome toxin-1 (TSST-1). Administration of the super antigen TSST-1 resulted in the specific systemic expansion of human V2+ T cells, release of human pro-inflammatory cytokines and localized specific activation and maturation of human CD11c+ dendritic cells. This novel mouse model of human hematopoiesis represents the first demonstration of long-term systemic human T cell reconstitution in vivo allowing for the manifestation of the differential response by human DCs to TSST-1.
Mucosal HIV Transmission
Human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS) is predominantly transmitted by unprotected sexual contact. Currently, women worldwide account for more than half of the estimated 11,000 newly acquired infections every day with a majority of those transmissions occurring via the vaginal route. Therefore, it is critical that strategies to prevent vaginal transmission of HIV are developed and implemented. However, of the estimated 341,524 male adults and adolescents living with HIV/AIDS in the US, 61% had been exposed through male-to-male sexual contact (HIV/AIDS Surveillance Report June 2007, CDC). Despite extensive educational campaigns promoting safe sexual practices, overall rates of HIV transmission due to Male-to-Male sexual contact increased by 13% between 2001 and 2005. These statistics clearly reflect an urgent need to devise and implement potential interventions that could prevent both vaginal and rectal HIV-1 transmission especially amongst high risk populations. The long term-goal of our laboratory is to investigate novel approaches to prevent HIV transmission. For this purpose we have developed and implemented a novel small animal model where human stem cells are used to reconstitute the hematopoietic system of immuno-deficient mice. In these humanized mice (designated BLT to represent the fact they are generated from a bone marrow transplant of mice previously implanted with a piece of autologous human fetal liver and thymic tissue) there is systemic reconstitution with human lymphoid cells in all hematopoietic and non-hematopoietic tissues tested. As we have previously shown and as it is illustrated in the Preliminary Results section, these humanized mice are susceptible to infection with HIV-1 administered either intra-rectally or intravaginally. Infection results in plasma antigenimia, development of anti-HIV specific human antibodies, progressive depletion of human CD4+ cells from the peripheral blood as well as systemic CD4 depletion especially in the gut associated lymphoid tissue (GALT). In this grant we propose to expand on these remarkable results and to address issues that are of fundamental importance to the rational develop-ment and implementation of pre-exposure prophylaxis measures to prevent HIV transmission.
Nef Function
The human immunodeficiency virus (HIV) types 1 and 2 and the simian immunodeficiency virus (SIV) are members of the lentivirus subfamily of the Retroviridae. They contain three genes (gag, pol, and env) that are present in all retroviruses and five (nef, rev, tat, vif, and vpr) that in combination are only present in HIV and SIV. HIV-1 also contains vpu that is not present in SIV or HIV-2. The latter six genes are referred to as accessory genes. However, this nomenclature is likely to minimize their importance because these proteins are essential virulence factors. The topic of this grant, nef, is found only in HIV and SIV where it is important for efficient virus replication and the eventual development of the pathology associated with AIDS in humans and primates. Therefore, though the biological significance of Nef has been proven, the exact function of Nef at the cellular level is not known. Nef alters intracellular trafficking of CD4 and MHC I. Nef also has a positive effect of virus infectivity. In addition, Nef alters signaling pathways in virus infected cells via its ability to activate Pak2. Although the molecular basis for these phenotypes remain to be fully elucidated, several important observations have been made that highlight the complex interplay between HIV and the infected cell. Nef might not be directly responsible for the development of the pathology associated with HIV disease but rather for the enhanced virus replication which results in the deterioration of the immune system of the infected host. One of the main challenges laying ahead is to correlate in vitro functions with the phenotypes observed in vivo. Progress made towards a better understanding of Nef function and its molecular determinants will facilitate the rational design of inhibitors of Nef function and of virus replication. Therefore, our work is focused on the molecular basis and consequences of the activation of Pak2 by Nef. Specifically we aim to:
1) Determine the molecular determinants of Pak2 activation by Nef.
2) Determine the role of Rho GTPases in the activation of Pak2 by Nef.
3) Determine the functional consequences of the activation of Pak2 by Nef.
4) Determine the role of Pak2 in HIV infection.
Hematopoietic Stem Cell Function
Hematopoietic stem cells (HSC) play a central role in the production of all hematopoietic lineages. HSC are functionally defined as having two important characteristics: self-renewal and the capacity to differentiate into all mature hematopoietic lineages. These two characteristics are in sharp contrast to those of mature cells that have limited proliferation and differentiation potential and lack self-renewal properties. In order to maintain a functional hematopoietic system, a population of HSC must be able to regenerate the entire hematopoietic repertoire on a regular basis. Whereas HSCs are readily evaluated by transplantation in mice, analysis in humans is quite difficult. Progress in the characterization of HSC has relied, for the most part, on in vitro assays such as clonogenic assays (CFCs) and of long-term cultures (LTC-IC). Interest in HSC is widespread because of their many possible clinical applications including transplantation, purging of tumor cells, and gene therapy. Recently, the use of xenografts has facilitated the analysis of the HSC. These models are based on the fact that human hematopoietic cells from fetal liver, bone marrow, umbilical cord or peripheral blood (after mobilization) can repopulate the bone marrow of severe combined immune deficient (SCID) mice. We are currently using two different but complementary xenograft models to evaluate the in vivo repopulating potential and the suitability for gene transfer of HSC from HIV infected individuals. Since ultimately this is the only source available for cells suitable genetic manipulations, this analysis represents an indispensable and important component of future clinical gene therapy protocols. Therefore we aim to:
1) Determine the quantitative in vivo repopulating potential of hematopoietic stem cells from HIV-1 infected individuals.
2) Determine the in vivo lymphopoietic capacity of T cell progenitors from HIV-1 infected individuals.
3) Characterize the primitive hematopoietic stem cell compartment in HIV-1 infected individuals.
Gene Therapy for AIDS
One highly significant weakness of most current systems to evaluate gene therapy approaches to AIDS is the lack of a reliable and robust in vivo system that could recapitulate all aspects of human T cell neogenesis. We tested the hypothesis that human hematopoietic stem cells (HSC) capable of repopulating long-term the bone marrow of recipient immune deficient mice could retain their full hematopoietic potential and give rise to T cell progenitors. Our results have fully validated this hypothesis. We have developed a novel small animal model that allows the in vivo evaluation of gene transfer into human T cell progenitors. In this system, HSC there is long-term in vivo repopulation of human, myeloid, B and T cell compartments. Furthermore, these animals also have readily detectable levels of plasmacytoid (CD123+) and myeloid (CD11c+) dendritic cells. Analysis of the T cell compartment in these humanized mice demonstrated the presence of single positive T cells as well as both naïve and memory T cells in the periphery, bone marrow and spleen. Furthermore, using lentivirus mediated gene transfer, we have shown that ex vivo transduced human HSC can engraft and give rise to all hematopoietic lineages while sustaining long-term transgene expression. In particular, we have shown transgene expression in both human thymocytes and T cells derived from ex vivo transduced human HSC. Now that we have established this system, we have proceeded with the next logical step of our studies: the in vivo characterization of transgene expression in the human T cell compartment in these humanized mice and the in vivo evaluation of molecular inhibitors of HIV replication.
Pre-Clinical Evaluation of Gene Therapy for Sickle Cell Disease
In recent years, the life expectancy and symptoms of patients with sickle cell disease (SCD) have improved significantly. These improvements have been due to clinical, genetic and molecular advances that have revealed basic aspects of the pathophysiology of SCD. In spite of these advances, SCD remains associated with significant mortality and morbidity. The large body of data with autologous stem cell bone marrow transplantation has shown to be effective for a certain number of patients with SCD but early mortality, the availability of suitable donors and patient selection remain limiting factors. Alternatively, genetic correction of SCD offers hope as a potential curative approach. Recent progress in the develop-ment of SCD mouse models and vector design have provided strong rational and impetus for preclinical implementation of gene therapy approaches for SCD. We are addressing three important challenges to the successful genetic correction of SCD. We first develop lentivirus-based vectors for the transduction of globin genes that include all critical regulatory elements for high level single copy gene expression. We then evaluate their transduction efficiency into stem cells from SC patients and finally we evaluate these transduced cells in vivo using a human/mouse xenograft model of bone marrow transplantation. The preclinical data obtained while conducting these experiments will serve as the rational basis for the implementation of clinical gene therapy protocols aimed at the genetic correction of sickle cell disease.
